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Coronavirus disease 2019 (COVID-19) is a highly transmissible disease that has affected more than 90% of the countries worldwide. At least 17 million individuals have been infected, and some countries are still battling first or second waves of the pandemic. Nucleic acid tests, especially reverse transcription polymerase chain reaction (RT-PCR), have become the workhorse for early detection of COVID-19 infection. Positive controls for the molecular assays have been developed to validate each test and to provide high accuracy. However, most available positive controls require cold-chain distribution and cannot serve as full-process control. To overcome these shortcomings, we report the production of biomimetic virus-like particles (VLPs) as SARS-CoV-2 positive controls. A SARS-CoV-2 detection module for RT-PCR was encapsidated into VLPs from a bacteriophage and a plant virus. The chimeric VLPs were obtained either by in vivo reconstitution and coexpression of the target detection module and coat proteins or by in vitro assembly of purified detection module RNA sequences and coat proteins. These VLP-based positive controls mimic SARS-CoV-2 packaged ribonucleic acid (RNA) while being noninfectious. Most importantly, we demonstrated that the positive controls are scalable, stable, and can serve broadly as controls, from RNA extraction to PCR in clinical settings.

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 Qβ 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.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

While highly promising in medicine, gene therapy requires delivery agents to protect and target nucleic acid therapeutics. We developed a plant viral siRNA delivery platform making use of self-assembling cowpea chlorotic mottle virus (CCMV). CCMV was loaded with siRNAs targeting GFP or FOXA1; to further enhance cell uptake and intracellular trafficking, resulting in more efficient gene knockdown, we appended CCMV with a cell penetrating peptide (CPP), specifically M-lycotoxin peptide L17E.

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.

Aim. The aim of the current work is to analyse the epiphytotic situation in the south of the Russian Far East in connection with viral diseases of legumes (Fabaceae Lindl., 1836).Discussion contains a description of 18 viruses that infect legumes in this region: Alfalfa mosaic (Martellivirales: Bromoviridae, Alfamovirus); Vicia unijuga mosaic (Martellivirales: Bromoviridae, Bromovirus); Cucumber mosaic (Martellivirales: Bromoviridae, Cucumovirus); Vicia unijuga ringspot virus (Martellivirales: Closteroviridae, Unidentified); Trifolium hybridum yellow mosaic virus, Bean common mosaic virus, Bean yellow mosaic virus, Trifolium repens mottle virus, Mountain clover mosaic virus, Red clover mosaic virus, Soybean chlorotic deformation virus, Soybean chlorotic mottle virus, Soybean mosaic virus, Soybean weak mosaic virus (Patatavirales: Genus, Potyvirus); Tobacco ringspot virus (Picornavirales: Secoviridae, Nepovirus); Pea enation mosaic virus (Tolivirales: Luteoviridae, Enamovirus); White clover mosaic virus (Tymovirales: Alphaflexiviridae, Potexvirus); Vicia pseudorobus necrotic mosaic virus (Tymovirales: Betaflexiviridae, Carlavirus). The description of the established natural reservoirs and the main vectors of these viruses is given.Conclusion. A list of measures are recommended for the prevention of viral diseases of legumes and a thesis is provided on the need to continue the planned monitoring of the phytovirological situation in the Russian Far East.

A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.

In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4(Trans) and Cp-Trans) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4(Rep) and Cp-Rep), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).

Virus crystals can incorporate a wide range of unusual impurities, not possible for conventional crystals, or even most protein crystals because of the large size of their constituent particles. These impurities include anomalous virions, satellite viruses and biological fibers. Examples of several of these unusual impurities are presented here, along with some of the consequences for the crystal lattices. The high solvent content, the forgiving character of the lattice and the plasticity of the virions allow these incorporations to be possible.

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

T=1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type T=3 virus with 1M CaCl2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 A resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 37 degrees , and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original T=3 BMV particles. The T=1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the T=1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the T=3 to T=1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and T=1 particles now permit us to propose a tentative model for that process. A comparison of the BMV T=1 particles was made with the reassembled T=1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 A, is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 A, has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.

The structure of brome mosaic virus (BMV), the type member of the bromoviridae family, has been determined from a single rhombohedral crystal by X-ray diffraction, and refined to an R value of 0.237 for data in the range 3.4-40.0 A. The structure, which represents the native, compact form at pH 5.2 in the presence of 0.1 M Mg(2+), was solved by molecular replacement using the model of cowpea chlorotic mottle virus (CCMV), which BMV closely resembles. The BMV model contains amino acid residues 41-189 for the pentameric capsid A subunits, and residues 25-189 and 1-189 for the B and C subunits, respectively, which compose the hexameric capsomeres. In the model there are two Mg ions and one molecule of polyethylene glycol (PEG). The first 25 amino acid residues of the C subunit are modeled as polyalanine. The coat protein has the canonical "jellyroll" beta-barrel topology with extended amino-terminal polypeptides as seen in other icosahedral plant viruses. Mass spectrometry shows that in native BMV virions, a significant fraction of the amino-terminal peptides are apparently cleaved. No recognizable nucleic acid residue is visible in the electron density maps except at low resolution where it appears to exhibit a layered arrangement in the virion interior. It is juxtaposed closely with the interior surface of the capsid but does not interpenetrate. The protein subunits forming hexameric capsomeres, and particularly dimers, appear to interact extensively, but the subunits otherwise contact one another sparsely about the 5-fold and quasi 3-fold axes. Thus, the virion appears to be an assembly of loosely associated hexameric capsomeres, which may be the basis for the swelling and dissociation that occurs at neutral pH and elevated salt concentration. A Mg ion is observed to lie exactly on the quasi-3-fold axis and is closely coordinated by side-chains of three quasi-symmetry-related residues glutamates 84, with possible participation of side-chains from threonines 145, and asparagines 148. A presumptive Mg(2+) is also present on the 5-fold axis where there is a concentration of negatively charged side-chains, but the precise coordination is unclear. In both cases these cations appear to be essential for maintenance of virion stability. Density that is contiguous with the viral interior is present on the 3-fold axis at the center of the hexameric capsomere, where there is a pore of about 6 A diameter. The density cannot be attributed to cations and it was modeled as a PEG molecule.

Brome mosaic virus (BMV), a T = 3 icosahedral plant virus, can be dissociated into coat protein subunits and subunit oligomers at pH 7.5 in the presence of concentrated salts. We have found that during the course of this treatment the coat protein subunits are cleaved, presumably by plant cell proteases still present in the preparation, between amino acids 35 and 36. The truncated protein subunits will then reorganize into T = 1 icosahedral particles and can be crystallized from sodium malonate. Quasi elastic light scattering and atomic force microscopy results suggest that the transition from T = 3 to T = 1 particles can occur by separate pathways, dissociation into coat protein subunits and oligomers and reassembly into T = 1 particles, or direct condensation of the T = 3 virions to T = 1 particles with the shedding of hexameric capsomeres. The latter process has been directly visualized using atomic force microscopy. Native T = 3 virions have been crystallized in several different crystal forms, but neither a rhombohedral form nor either of two orthorhombic forms diffract beyond about 3.4 A. Tetragonal crystals of the T = 1 particles, however, diffract to at least 2.5 A resolution. Evidence suggests that the T = 1 particles are more structurally uniform and ordered than are native T = 3 virions. A variety of anomalous virus particles having diverse sizes have been visualized in preparations of BMV used for crystallization. In some cases these aberrant particles are incorporated into growing crystals where they are frequently responsible for defect formation.

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

BackgroundRNA-protein interactions stabilize many viruses and also the nucleoprotein cores of enveloped animal viruses (e.g. retroviruses). The nucleoprotein particles are frequently pleomorphic and generally unstable due to the lack of strong protein-protein interactions in their capsids. Principles governing their structures are unknown because crystals of such nucleoprotein particles that diffract to high resolution have not previously been produced. Cowpea chlorotic mottle virions (CCMV) are typical of particles stabilized by RNA-protein interactions and it has been found that crystals that diffract beyond 4.5 A resolution are difficult to grow. However, we report here the purification of CCMV with an exceptionally mild procedure and the growth of crystals that diffract X-rays to 3.2 A resolution.ResultsThe 3.2 A X-ray structure of native CCMV, an icosahedral (T = 3) RNA plant virus, shows novel quaternary structure interactions based on interwoven carboxyterminal polypeptides that extend from canonical capsid beta-barrel subunits. Additional particle stability is provided by intercapsomere contacts between metal ion mediated carboxyl cages and by protein interactions with regions of ordered RNA. The structure of a metal-free, swollen form of the virus was determined by cryo-electron microscopy and image reconstruction. Modeling of this structure with the X-ray coordinates of the native subunits shows that the 29 A radial expansion is due to electrostatic repulsion at the carboxyl cages and is stopped short of complete disassembly by preservation of interwoven carboxyl termini and protein-RNA contacts.ConclusionsThe CCMV capsid displays quaternary structural interactions that are unique compared with previously determined RNA virus structures. The loosely coupled hexamer and pentamer morphological units readily explain their versatile reassembly properties and the pH and metal ion dependent polymorphism observed in the virions. Association of capsomeres through inter-penetrating carboxy-terminal portions of the subunit polypeptides has been previously described only for the DNA tumor viruses, SV40 and polyoma.

Toll-like receptors (TLRs) are promising targets in cancer immunotherapy due to their role in activating the immune system; therefore, various small-molecule TLR agonists have been tested in clinical applications. However, the clinical use of TLR agonists is hindered by their non-specific side effects and poor pharmacokinetics. To overcome these limitations, we used plant virus nanoparticles (VNPs) and bacteriophage virus-like particles (VLPs) as drug delivery systems. We conjugated TLR3 or TLR7 agonists to cowpea mosaic virus (CPMV) VNPs, cowpea chlorotic mottle virus (CCMV) VNPs, and bacteriophage Qβ VLPs. The conjugation of TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), resulted in the potent activation of immune cells and promoted the production of pro-inflammatory cytokine interleukin 6. We found that 1V209 conjugated to CPMV, CCMV, and Qβ reduced tumor growth in vivo and prolonged the survival of mice compared to those treated with free 1V209 or a simple admixture of 1V209 and viral particles. Nucleic acid-based TLR3 agonist, polyinosinic acid with polycytidylic acid (poly(I:C)), was also delivered by CPMV VNPs, resulting in enhanced mice survival. All our data suggest that coupling and co-delivery are required to enhance the anti-tumor efficacy of TLR agonists and simple mixing of the VLPs with the agonists does not confer a survival benefit. The delivery of 1V209 or poly(I:C) conjugated to VNPs/VLPs probably enhances their efficacy due to the multivalent presentation, prolongation of tumor residence time, and targeting of the innate immune cells mediated by the VNP/VLP carrier.

Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable such observations, we have recently proposed a technique that improves the time resolution of cryo-electron microscopy (cryo-EM) to microseconds. Here, we demonstrate that microsecond time-resolved cryo-EM enables observations of fast protein dynamics. We use our approach to elucidate the mechanics of the capsid of cowpea chlorotic mottle virus (CCMV), whose large-amplitude motions play a crucial role in the viral life cycle. We observe that a pH jump causes the extended configuration of the capsid to contract on the microsecond timescale. While this is a concerted process, the motions of the capsid proteins involve different timescales, leading to a curved reaction path. It is difficult to conceive how such a detailed picture of the dynamics could have been obtained with any other method, which highlights the potential of our technique. Crucially, our experiments pave the way for microsecond time-resolved cryo-EM to be applied to a broad range of protein dynamics that previously could not have been observed. This promises to fundamentally advance our understanding of protein function., (© 2023. Springer Nature Limited.)

We extend a recently proposed kinetic theory of virus capsid assembly based on Model A kinetics and study the dynamics of the interconversion of virus capsids of different sizes triggered by a quench, that is, by sudden changes in the solution conditions. The work is inspired by in vitro experiments on functionalized coat proteins of the plant virus cowpea chlorotic mottle virus, which undergo a reversible transition between two different shell sizes (T = 1 and T = 3) upon changing the acidity and salinity of the solution. We find that the relaxation dynamics are governed by two time scales that, in almost all cases, can be identified as two distinct processes. Initially, the monomers and one of the two types of capsids respond to the quench. Subsequently, the monomer concentration remains essentially constant, and the conversion between the two capsid species completes. In the intermediate stages, a long-lived metastable steady state may present itself, where the thermodynamically less stable species predominate. We conclude that a Model A based relaxational model can reasonably describe the early and intermediate stages of the conversion experiments. However, it fails to provide a good representation of the time evolution of the state of assembly of the coat proteins in the very late stages of equilibration when one of the two species disappears from the solution. It appears that explicitly incorporating the nucleation barriers to assembly and disassembly is crucial for an accurate description of the experimental findings, at least under conditions where these barriers are sufficiently large., (© 2023 Author(s). Published under an exclusive license by AIP Publishing.)

Ecological strategies for resource utilisation are important features of pathogens, yet have been overshadowed by stronger interest in genetic mechanisms underlying disease emergence. The purpose of this study is to ask whether host range and transmission traits translate into ecological strategies for host-species utilisation in a heterogeneous ecosystem, and whether host utilisation corresponds to genetic differentiation among three bromoviruses. We combine high-throughput sequencing and population genomics with analyses of species co-occurrence to unravel the ecological strategies of the viruses across four habitat types. The results show that the bromoviruses that were more closely related genetically did not share similar ecological strategies, but that the more distantly related pair did. Shared strategies included a broad host range and more frequent co-occurrences, which both were habitat-dependent. Each habitat thus presents as a barrier to gene flow, and each virus has an ecological strategy to navigate limitations to colonising non-natal habitats. Variation in ecological strategies could therefore hold the key to unlocking events that lead to emergence., Competing Interests: The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

Background: Members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis E virus and rubella virus. They encode a capping enzyme with methyltransferase-guanylyltransferase (MTase-GTase) activity, which is an attractive drug target owing to its unique mechanism. However, its experimental study has proven very difficult. Results: We examined over 50 genera of viruses by sequence analyses. Earlier studies showed that the MTase-GTase contains a "Core" region conserved in sequence. We show that it is followed by a long extension, which we termed "Iceberg" region, whose secondary structure, but not sequence, is strikingly conserved throughout the alphavirus supergroup. Sequence analyses strongly suggest that the minimal capping domain corresponds to the Core and Iceberg regions combined, which is supported by earlier experimental data. The Iceberg region contains all known membrane association sites that contribute to the assembly of viral replication factories. We predict that it may also contain an overlooked, widely conserved membrane-binding amphipathic helix. Unexpectedly, we detected a sequence homolog of the alphavirus MTase-GTase in taxa related to nodaviruses and to chronic bee paralysis virus. The presence of a capping enzyme in nodaviruses is biologically consistent, since they have capped genomes but replicate in the cytoplasm, where no cellular capping enzyme is present. The putative MTase-GTase domain of nodaviruses also contains membrane-binding sites that may drive the assembly of viral replication factories, revealing an unsuspected parallel with the alphavirus supergroup. Conclusions: Our work will guide the functional analysis of the alphaviral MTase-GTase and the production of domains for structure determination. The identification of a homologous domain in a simple model system, nodaviruses, which replicate in numerous eukaryotic cell systems (yeast, flies, worms, mammals, and plants), can further help crack the function and structure of the enzyme. [ABSTRACT FROM AUTHOR]